1. Field of the Invention
The present invention relates to lateral flow test strips and methods of operation for the lateral flow test strips.
2. Description of Related Art
Quantitative analysis of cells and analytes in fluid samples, particularly bodily fluid samples, often provides critical diagnostic and treatment information for physicians and patients. For example, immunological testing methods which take advantage of the high specificity of antigen-antibody reactions, provide one approach to measurement of analytes. Kennedy, D. M. and S. J. Challacombe, eds., ELISA and Other Solid Phase Immunoassays: Theoretical and Practical Aspects, John Wiley and Sons, Chichester (1988). This document and all others cited to herein, are incorporated by reference as if reproduced fully below. Such assays may also find use in various other applications, such as veterinary, food testing, or agricultural applications.
Immunoassays that provide a quantitative measurement of the amount of an analyte in a sample have previously used complex, multi-step procedures and expensive analyzers available only in a laboratory setting.
Immunochromatographic assays, such as those described in GB 2,204,398A; U.S. Pat. Nos. 5,096,837, 5,238,652, and 5,266,497; Birnbaum, S. et al., Analytical Biochem. 206:168-171 (1992); Roberts, M. A. and R. A. Durst, Analytical Chem. 67:482-491 (1995); and Klimov, A. D. et al., Clinical Chem. 41:1360 (1995), are simpler, yet do not provide a quantitative measurement of an analyte. Instead, these immunochromatographic assays detect the presence (or absence) of an analyte above a defined cutoff level the test performed. The lack of a quantitative measurement limits the usefulness of these assays.
A variety of disposable diagnostic assay devices have also been developed. Examples of such devices include, but are not limited to Cathey, et al, U.S. Pat. No. 5,660,993; International Publication Number WO 92/12428; Eisinger, et al, U.S. Pat. No. 4,943,522; Campbell, et al, U.S. Pat. No. 4,703,017; Campbell, et al, U.S. Pat. No. 4,743,560; and Brooks, U.S. Pat. No. 5,753,517.
A test strip is provided which is adapted to receive a sample and detect an analyte therein. According to one embodiment, the test strip comprises a sample addition zone to which a sample may be added; an absorbent zone proximal to the sample addition zone; one or more test zones distal to the sample addition zone, at least one of the test zones including a first analyte binding agent immobilized therein which is capable of binding to the analyte to be detected; and a terminal sample flow zone distal to the one or more test zones, the absorbent zone being positioned relative to the sample addition zone and having an absorption capacity relative to the other zones of the test strip such that a distal diffusion front of a sample added to the sample addition zone diffuses from the sample addition zone to a distal diffusion point within the terminal sample flow zone and then reverses direction and diffuses proximal relative to the one or more test zones.
In another embodiment, a test strip is provided which comprises a sample addition zone to which a sample may be added; an absorbent zone proximal to the sample addition zone; one or more test zones distal to the sample addition zone, at least one of the test zones including a first analyte binding agent immobilized therein which is capable of binding to the analyte to be detected; a terminal sample flow zone distal to the one or more test zones, the absorbent zone being positioned relative to the sample addition zone and having an absorption capacity relative to the other zones of the test strip such that a distal diffusion front of a sample added to the sample addition zone within the predetermined volume range diffuses from the sample addition zone to a distal diffusion point within the terminal sample flow zone and then diffuses proximal relative to the one or more test zones; and a conjugate buffer addition zone distal to the terminal sample flow zone to which a conjugate buffer may be added.
According to the above test strip embodiment, the conjugate buffer addition zone may be positioned relative to the test zones such that conjugate buffer added to the conjugate buffer addition zone at the same time as sample is added to the sample addition zone reaches the distal diffusion point after the distal diffusion front of the sample has diffused to the distal diffusion point and begun diffusing in a proximal direction. The conjugate buffer addition zone may also be positioned relative to the test zones such that conjugate buffer added to the conjugate buffer addition zone at the same time that the sample is added to the sample addition zone reaches the test zones after the distal diffusion front of the sample diffuses proximal relative to the test zones. The conjugate buffer addition zone may also be positioned relative to the test zones such that the conjugate buffer can be added to the test strip before the sample and nevertheless reach the distal diffusion point after the distal diffusion front of the sample has diffused to the distal diffusion zone, reversed direction and begun diffusing in a proximal direction.
According to any of the above test strip embodiments, the test strip may include 1, 2, 3 or more test zones with one or more control binding agents immobilized therein. In one embodiment, the test strip comprises at least a first control zone with a control binding agent immobilized therein. Optionally, the test zones further include a second control zone with a same control binding agent immobilized therein as the first control zone, the first control zone containing a different amount of the control binding agent than the second control zone.
Also according to any of the above test strip embodiments, a second analyte binding agent which is capable of binding to the analyte and diffusing to the one or more test zones may be included on the test strip. Alternatively, second analyte binding agent may be delivered to the test strip via the conjugate buffer. The second analyte binding agent may bind to components in the sample other than the analyte. Alternatively, the second analyte binding agent may be an agent which does not bind to components in the sample other than the analyte.
In order to facilitate detection, the second analyte binding agent is preferably labeled with a detectable marker. As discussed herein, any of a wide range of detectable markers known in the art may be used. In a preferred embodiment, the second analyte binding agent is attached to a particle which is capable of diffusing to the one or more test zones. The particle may serve as the detectable marker or may itself be labeled with a detectable marker.
A method is also provided for detecting an analyte in a sample. In one embodiment, the method comprises delivering a sample to a test strip which causes a distal diffusion front of the sample to (a) diffuse in a distal direction to one or more test zones, at least one of the test zones including a first analyte binding agent immobilized therein which binds to analyte in the sample, (b) diffuse to a terminal sample flow zone distal to the one or more test zones, change direction and (c) diffuse to a position proximal to the one or more test zones; delivering a conjugate buffer to the test strip at a position distal to the terminal sample flow zone, delivery of the conjugate buffer causing a second analyte binding agent to diffuse proximally past the terminal sample flow zone to the one or more test zones after the distal diffusion front of the sample diffuses proximal to the one or more test zones, the second analyte binding agent binding to analyte immobilized in the test zones; and detecting the second analyte binding agent immobilized in the test zones.
According to the method, the conjugate buffer may be added to the test strip at a same time as the sample is added to the test strip, before the sample is added to the test strip, or after the sample is added to the test strip. When the sample is added to the test strip relative to the conjugate buffer depends on the time required for the sample to reach the terminal sample flow zone which, in turn, depends on the flow design of the test strip.
Also according to the method, the second analyte binding agent may be contained on the test strip where the conjugate buffer is delivered, delivery of the conjugate buffer causing the diffusion of the second analyte binding agent. Alternatively, the second analyte binding agent is contained on the test strip proximal to where the conjugate buffer is delivered, delivery of the conjugate buffer causing the diffusion of the second analyte binding agent. Delivering the conjugate buffer to the test strip may also include delivering the second analyte binding agent to the test strip within the conjugate buffer.
According to the above method, the test zones may include a first control zone with a control binding agent immobilized therein, delivering the conjugate buffer causing a control agent to diffuse proximally past the terminal sample flow zone to the first control zone and bind to the control binding agent immobilized therein. Alternatively, the test zones may include first and second control zones which each include a different amount of a control binding agent immobilized therein, delivering the conjugate buffer causing a control agent to diffuse proximally past the terminal sample flow zone to the first and second control zones and bind to the control binding agent immobilized therein.
Also according to the above method, detecting the second analyte binding agent may be facilitated by labeling the second analyte binding agent with a detectable marker, detecting the second analyte binding agent including detecting the detectable marker. The second analyte binding agent may be attached to a particle. Detecting the second analyte binding agent may include detecting the particle.
According to any of the above embodiments, the sample delivered to the test strip is preferably within a predetermined volume range that the test strip has been designed to process. The predetermined volume range is preferably between about 10 and 250 xcexcL, preferably between about 20 and 100 xcexcL, more preferably between about 30 and 50 xcexcL, and most preferably between about 35 and 45 xcexcL. When a sample is delivered to the test strip within the predetermined volume range, the terminal sample flow zone may be designed to have a short length from a proximal end to a distal end. For example, when a sample is delivered to the test strip within a range of about 35 and 45 xcexcL, the terminal sample flow zone may have a length from a proximal end to a distal end of between about 1 and 25 mm, more preferably 2 and 15 mm, and most preferably 3 and 10 mm.
Also according to any of the above embodiments, the first analyte binding agent preferably does not bind to components in the sample other than the analyte. Types of molecules that can serve as first analyte binding agents include, but are not limited to antibodies, engineered proteins, peptides, haptens, lysates containing heterogeneous mixtures of antigens having analyte binding sites, ligands and receptors. In one particular embodiment, the first analyte binding agent is an antibody or fragment thereof.